Nucleic acids encoding an anti-CD154 antibody comprising E269R and K322A mutations

ABSTRACT

Improved anti-CD154 antibodies are provided herein which have ablated FcR binding and/or complement binding/activation. The use of these antibodies for inducing tolerance and treating immune diseases including autoimmunity, inflammation and allergic disorders is disclosed herein.

PRIORITY INFORMATION Related Applications

The present application is a divisional of U.S. Ser. No. 15/657,837filed Jul. 24, 2017, which is a divisional of U.S. Ser. No. 14/681,905,filed Apr. 8, 2015 (now U.S. Pat. No. 9,758,587), which is acontinuation of U.S. Ser. No. 13/798,601 filed Mar. 13, 2013 (now U.S.Pat. No. 9,028,826), which is a continuation-in-part of U.S. Ser. No.13/648,808 filed Oct. 10, 2012 (now U.S. Pat. No. 9,321,833), which is acontinuation-in-part of U.S. Ser. No. 13/439,186, filed on Apr. 4, 2012(now U.S. Pat. No. 8,852,597), which claims priority to provisionalapplication U.S. Ser. No. 61/471,287 filed on Apr. 4, 2011. All of theseapplications are incorporated by reference in their entirety.

SEQUENCE LISTING

The sequence listing in the file named “1143260o000110.txt” having asize of 50,084 bytes that was created Oct. 15, 2020, is herebyincorporated by reference in its entirety.

BACKGROUND Field of the Invention

The present invention relates to improved anti-CD154 (CD40L) antibodieshaving reduced toxicity and their use in immune therapies, especiallytreatment of inflammatory disorders, allergy, autoimmunity, transplant,and cancers. In particular the invention provides anti-CD154 antibodiesthat are modified such that they do not elicit thrombogenic or clottingreactions in vivo, but which still retain desired therapeutic propertiessuch as the induction of immune tolerance and the blockade of humoralimmunity.

Description of Related Art

CD40L (CD154) is a highly validated and valuable therapeutic target inautoimmunity, graft rejection and other immune-related diseases in mice,non-human primates (NHP) and humans. In numerous Phase II ClinicalTrials, α-CD154 has been shown to effectively block the activities ofCD154 in vivo and ameliorate disease. αCD154 is distinct from all othertherapeutics in its impact on the immune response; it is one of the onlytherapeutics that can induce functional immunological tolerance, asdemonstrated both in mice and monkeys. In mice, virtually all autoimmunedisease models can be effectively ameliorated with αCD154 therapy(Noelle, R. J., Mackey, M., Foy, T., Buhlmann, J. and Burns, C., CD40and its ligand in autoimmunity. Ann N Y Acad Sci 1997. 815: 384-391;Mackey, M. F., Barth, R. J., Jr. and Noelle, R. J., The role ofCD40/CD154 interactions in the priming, differentiation, and effectorfunction of helper and cytotoxic T cells. J Leukoc Biol 1998. 63:418-428; Noelle, R. J., CD40 and its ligand in cell-mediated immunity.Agents Actions Suppl 1998. 49: 17-22; and Quezada, S. A., Jarvinen, L.Z., Lind, E. F. and Noelle, R. J., CD40/CD154 Interactions at theInterface of Tolerance and Immunity. Annu Rev Immunol 2004. 22:307-328), with long-term remission observed.

In NHP, permanent allograft tolerance can be achieved using shortcourses of treatments comprised of αCD154 (Kenyon, N. S., Chatzipetrou,M., Masetti, M., Ranuncoli, A., Oliveira, M., Wagner, J. L., Kirk, A.D., Harlan, D. M., Burkly, L. C. and Ricordi, C., Long-term survival andfunction of intrahepatic islet allografts in rhesus monkeys treated withhumanized anti-CD154. Proc Natl Acad Sci USA 1999. 96: 8132-8137; Kirk,A. D., Burkly, L. C., Batty, D. S., Baumgartner, R. E., Berning, J. D.,Buchanan, K., Fechner, J. H., Jr., Germond, R. L., Kampen, R. L.,Patterson, N. B., Swanson, S. J., Tadaki, D. K., TenHoor, C. N., White,L., Knechtle, S. J. and Harlan, D. M., Treatment with humanizedmonoclonal antibody against CD154 prevents acute renal allograftrejection in nonhuman primates. Nat Med 1999. 5: 686-693).

Also, Phase II Clinical Trials in humans have indicated that αCD154 iseffective in SLE (Sidiropoulos, P. I. and Boumpas, D. T., Lessonslearned from anti-CD154 treatment in systemic lupus erythematosuspatients. Lupus 2004. 13: 391-397), Multiple Sclerosis (see preliminarydata) and idiopathic thrombocytopenia (Sidiropoulos, P. I. and Boumpas,D. T., Lessons learned from anti-CD154 treatment in systemic lupuserythematosus patients. Lupus 2004. 13: 391-39). As such, αCD154 is aunique drug that will allow for short-term intervention with long-termclinical benefit. Its failures have not been in efficacy, but due to anunanticipated toxicity.

Further, in the early 1990's IDEC Pharmaceuticals and Biogen Inc. (nowBiogen Idec) launched two different αCD154 mAbs into multiple Phase I/IIClinical Trials. The antibody developed by IDEC (IDEC-131) was derivedfrom a murine anti-hCD154 developed at Dartmouth College.

This antibody and humanized variants are disclosed in U.S. Pat. No.6,440,418 the contents of which are incorporated by reference herein.While early indications demonstrated that the drug was highly effective,toxicity of the αCD154 prohibited continued clinical development. In thetrials, the observed toxicity included the induction of thromboembolicevents in patients. Based on toxicity concerns, all trials weresuspended and efforts were directed towards re-engineering the mAbs tosustain efficacy and reduce toxicity. While reduced toxicity has beenachieved, there has been a substantial decrease in efficacy and thetolerance-inducing capacity of αCD154 mAbs (Ferrant, J. L., Benjamin, C.D., Cutler, A. H., Kalled, S. L., Hsu, Y. M., Garber, E. A., Hess, D.M., Shapiro, R. I., Kenyon, N. S., Harlan, D. M., Kirk, A. D., Burkly,L. C. and Taylor, F. R., The contribution of Fc effector mechanisms inthe efficacy of anti-CD154 immunotherapy depends on the nature of theimmune challenge. Int Immunol 2004. 16: 1583-1594). None of theengineered mAb forms have progressed significantly into the clinic dueto loss in efficacy.

Recently, Biogen-Idec and UCB entered into a collaboration relating tothe study of anti-CD40L antibodies (see,http://www.news-medical.net/news/20111219/ALS-TDI-Biogen-Idec-and-UCB-enter-agreement-to-study-anti-CD40L-antibody-for-ALS.aspx).This study relates to a Pegylated Fab of an αCD154.

Notwithstanding the foregoing, there is still a significant need in theart for improved anti-CD154 antibodies, i.e., those which are both safeand effective. This invention attains these goals.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 shows the impact of anti-CD154 therapy on relapse rate in RR MSPatients. Patients with active disease were recruited into trial andwere treated with 4 escalating doses of IDEC-131 every week. Followingtreatment all patients were followed by EDSS scoring as well asassessment of gadolinium-enhanced lesions.

FIG. 2 shows the nucleotide sequence of a hamster anti-murine CD154.Shown are the k and heavy chain sequence for the MR1 anti-human CD40L(CD154) hamster IgG1.

FIG. 3 shows the reduction in FcR binding in the E223PIgG1 MR1 IgG1variant.

FIG. 4 shows the effects of mutations in MR1 that ablate C1q binding.

FIG. 5 shows that the loss of complement activation does not reduce theability of anti-CD154 to induce tolerance.

FIG. 6 shows the thrombotic stress signs in all tested animals arrangedby treatment groups.

FIG. 7 shows platelet counts of all animals used in the study, arrangedby treatment groups.

FIG. 8 : Average number of clots per field (200× original magnification)as observed microscopically, arranged by treatment groups.

FIG. 9 : Sample images of H&E stained lung sections from animalsinjected with PBS. Header values indicate original microscopicmagnification. With these example images and with those below, thehigher magnification images were acquired from within the field of thefirst (100×) image.

FIG. 10 : Sample images of H&E stained lung sections from animalsinjected with MR1-WT. Header values indicate original microscopicmagnification. Blue arrow identifies thrombus.

FIG. 11 : Sample images of H&E stained lung sections from animalsinjected with N325L. Header values indicate original microscopicmagnification

FIG. 12 : Sample images of H&E stained lung sections from animalsinjected with K326V. Header values indicate original microscopicmagnification

FIG. 13 : Sample images of H&E stained lung sections from animalsinjected with E269R. Header values indicate original microscopicmagnification.

FIGS. 14-16 contain humanized sequences corresponding to an anti-humanCD154 antibody (IDEC-131).

FIGS. 17 and 18 contain the variable sequences for the parent chimericantibody from which IDEC-131 was derived.

FIG. 19 contains ELISA hCD40L binding data for two anti-human CD40L IgG1antibodies according to the invention, Antibody A and antibody B whichwere expressed in HEK-293 Freestyle cells, wherein each antibodycomprises the variable heavy and light sequences in FIG. 20 andmodifications in the human IgG1 constant region. Antibody A containsV215A and E269R mutations in the constant region. Antibody B contains 3mutations in the constant region, i.e., V215A, E269R and K322Amutations.

FIG. 20 contains the variable heavy and light sequences and constantregion for an antibody according to the invention.

FIG. 21 contains an alignment of antibody sequences including anti-CD40Lantibody sequences according to the invention.

DETAILED DESCRIPTION

Prior to disclosing the invention in detail the following definitionsare provided. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art to which this invention belongs.

As used herein, oligonucleotide sequences that are complementary to oneor more of the genes described herein, refers to oligonucleotides thatare capable of hybridizing under stringent conditions to at least partof the nucleotide sequence of said genes. Such hybridizableoligonucleotides will typically exhibit at least about 75% sequenceidentity at the nucleotide level to said genes, preferably about 80% or85% sequence identity or more preferably about 90% or 95% or moresequence identity to said genes.

“Bind(s) substantially” refers to complementary hybridization between aprobe nucleic acid and a target nucleic acid and embraces minormismatches that can be accommodated by reducing the stringency of thehybridization media to achieve the desired detection of the targetpolynucleotide sequence.

The phrase “hybridizing specifically to” refers to the binding,duplexing or hybridizing of a molecule substantially to or only to aparticular nucleotide sequence or sequences under stringent conditionswhen that sequence is present in a complex mixture (e.g., totalcellular) DNA or RNA.

“Mutation or mutations that eliminate or reduces FcR binding and whicheliminates toxicity” herein refers to a mutation or mutations thateliminate or reduce thrombocytopenia or thrombosis or clotting in vivo.The efficacy of such mutations in eliminating or reducingthrombocytopenia or thrombosis or clotting is shown infra murinethrombosis model, e.g., a mouse that has been engineered to expresshuman FcR, and wherein said mouse is administered a chimeric antibodyspecific to a host compatible, i.e., anti-rodent CD40L, e.g., mouseCD40L, and which chimeric antibody contains either human IgG1 or IgG3constant regions, and further wherein the Fc region thereof has beenmutated at one or more sites to eliminate or substantially inhibit FcRbinding. Exemplary sites which may be modified and specific mutationsare identified in Tables 2-4 and the examples infra.

“Mutation or mutations that eliminate or reduce complement function andwhich maintain tolerance inducing properties” refers to mutation ormutations in human, chimeric, or humanized antibodies containing humanconstant regions, preferably human IgG1 or IgG3 constant regions,wherein the Fc region thereof has been mutated at one or more sites inorder to eliminate or substantially reduce complement binding.Preferably such mutations will not appreciably affect the ability of theantibody to induce tolerance in vivo. This may be established usingappropriate tolerance models such as the skin transplant model disclosedherein. Exemplary sites which may be modified and specific mutations areidentified in Tables 2-4 and the examples infra.

A “patient” can mean either a human or non-human animal, preferably amammal. In preferred embodiments this invention produces anti-humanCD40L antibodies suitable for human therapy containing mutated IgG1 orIgG3 constant regions, wherein such mutations eliminate or substantiallyinhibit toxicity concerns such as thrombocytopenia or thrombosis orclotting reactions or toxicity associated with complement reactions andpreferably wherein such antibodies retain the ability to inducetolerance or humoral suppression in vivo.

As used herein, “subject”, as refers to an organism or to a cell sample,tissue sample or organ sample derived therefrom, including, for example,cultured cell lines, biopsy, blood sample, or fluid sample containing acell. In many instances, the subject or sample derived therefrom,comprises a plurality of cell types. In one embodiment, the sampleincludes, for example, a mixture of tumor and normal cells. In oneembodiment, the sample comprises at least 10%, 15%, 20%, et seq., 90%,or 95% tumor cells. The organism may be an animal, including but notlimited to, an animal, such as a cow, a pig, a mouse, a rat, a chicken,a cat, a dog, etc., and is usually a mammal, such as a human.

The term “treating” in its various grammatical forms in relation to thepresent invention refers to preventing (i.e. chemoprevention), curing,reversing, attenuating, alleviating, minimizing, suppressing, or haltingthe deleterious effects of a disease state, disease progression, diseasecausative agent (e.g. bacteria or viruses), or other abnormal condition.For example, treatment may involve alleviating a symptom (i.e., notnecessarily all the symptoms) of a disease of attenuating theprogression of a disease.

“Treatment of autoimmunity” or “treating” another disease condition suchas cancer, infection, allergy, transplant, graft versus host disease andother conditions wherein anti-CD154 antibodies are potentially oftherapeutic benefit as used herein, refers to partially or totallyinhibiting, delaying, or preventing the progression of the disease. Inthe case of cancer this means treating or inhibiting cancer metastasis;inhibiting, delaying, or preventing the recurrence of cancer includingcancer metastasis; or preventing the onset or development of cancer(chemoprevention) in a mammal, for example, a human. In addition, themethods of the present invention may be practiced for the treatment ofhuman patients with cancer. However, it is also likely that the methodswould also be effective in the treatment of cancer in other mammals. Inthe preferred embodiments the subject antibodies are used to treatautoimmunity, allergy, inflammation, transplant, GVHD, bone marrowtransplant (BMT), and to induce antigen specific tolerance in subjectsin need thereof. Preferred indications are multiple sclerosis, lupus,ITP, IBD, Crohn's disease, psoriasis, uveitis, rheumatoid arthritis,asthma, GVHD, bone marrow transplant, oophoritis and thyroiditis.

As used herein, the term “therapeutically effective amount” is intendedto qualify the amount of the treatment in a therapeutic regimentnecessary to treat a condition e.g., autoimmunity.

The present invention provides novel and improved anti-CD154 antibodiesfor use in therapies containing mutated Fc regions that are safe andeffective. These antibodies exhibit improved safety and efficacycompared to currently available anti-CD154 antibodies because ofmutations that inhibit or prevent FcR binding and/or complement bindingand activation.

In especially preferred embodiments the subject anti-CD154 antibodieswill comprise the same CDR's as IDEC 131 and more specifically containthe humanized variable heavy and light regions of IDEC-131 or willcontain a humanized variable light region and a variable heavy chainregion that is at least 95%, 98% or which is identical to at least oneof the humanized variable light and variable heavy sequences identifiedas (1), (2), (3) and (4) set forth below.

In these variable heavy and light amino acid sequences which are setforth below, the FR and CDR regions are separated by spaces and thesequences are organized as in normal antibody variable regions, i.e.:FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4.

For example, in variable light sequence (1) the CDRs of the variablelight chain are KASQNVITAVA (CDR1), SASNRYT (CDR2) and QQYNSYPYT (CDR3)and the CDRs of the heavy chain sequence (1) are respectively NGFWI(CDR1), YISYSGSTYYNPSLKS (CDR2) and RSYGRTPYYFDF (CDR3). In preferredembodiments the anti-CD154 antibody will contain a variable light and avariable heavy sequence identical to one of those identified as variablelight and variable heavy sequences (1) and (2) (any combination thereof)and the Fc region of the humanized anti-CD154 antibody will comprisemutations in the Fc region, e.g., a human IgG1 or IgG3 constant region,that preclude one or both of FcR binding and complement binding, andwill be safe (not elicit thrombogenic or clotting reaction) and willinduce immune tolerance or suppression of humoral immunity in vivo.

The preferred humanized variable light sequences (1), (2) (3) and (4)are forth below:

(1) DIVMTQSPSFLSASVGDRVTITC KASQNVITAVA WYQQKPGKSPKLLIY SASNRYT GVPDRFSGSGSGTDFTLTISSLQPEDFADY FC QQYNSYPYT FGGGTKLEIK;(2) DIVMTQSPDSLAVSLGERATINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT GVPDRFSGSGSGTDFTLTISSLQAEDVADY FC QQYNSYPYT FGGGTKLEIK;(3) DIVMTQSPSFMSTSVGDRVTITC KASQNVITAVA WYQQKPGKSPKLLIY SASNRYT GVPDRFSGSGSGTDFTLTISSMQPEDFADY FC QQYNSYPYT FGGGTKLEIK;(4) DIVMTQSPDSMATSLGERVTINC KASQNVITAVA WYQQKPGQSPKLLIY SASNRYT GVPDRFSGSGSGTDFTLTISSMQAEDVADY FC QQYNSYPYT. FGGGTKLEIK,

The preferred humanized variable heavy sequences (1), (2) (3) and (4)are forth below:

(1) EVQLQESGPGLVKPSETLSLTCTVSGDSIT NGFWI WIRKPPGNKLEYMG YISYSGSTYYNPSLKS RISISRDTSKNQFSLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS; (2)EVQLQESGPGLVKPSQTLSLTCTVSGDSIT NGFWI WIRKHPGNKLEYMG YISYSGSTYYNPSLKS RISISRDTSKNQFSLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS; (3)EVQLQESGPGLVKPSQTLSLTCAVSGDSIT NGFWI WIRKHPGNKLEYMG YISYSGSTYYNPSLKS RISISRDTSNNQFSLNLNSVTRADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS; (4)EVQLQESGPGLVKPSETLSLTCAVYGDSIT NGFWI WIRKPPGNKLEYMG YISYSGSTYYNPSLKS RISISRDTSKNQFYLKLSSVTAADTGVYYCAC RSYGRTPYYFDF WGQGTTLTVSS.

As discussed, the subject antibodies preferably will be mutated toinhibit or prevent complement binding and complement activation as wellas mutations that preclude or which substantially inhibit FcR binding.This mutation was not anticipated to be beneficial to an anti-CD154antibody to be used as a therapeutic, as prior to this invention it wasthought that the therapeutic efficacy of an αCD154, aside from itsability to simply block CD154, also required complement binding. It wasthought by experts that complement binding was critical for CD154antibodies to actively induce tolerance. Also, prior to this inventionit was uncertain as to whether the ability of the αCD154 antibody tobind FcR would potentially adversely affect therapeutic functionality,i.e., the antibody's ability to induce tolerance.

Notwithstanding the foregoing, the inventor proposed to develop mutatedanti-CD154 antibodies that do not bind FcR and/or complement with thehope that such antibodies would maintain the full tolerance-inducingcapacity of αCD154, while eliminating its toxicity. Such an antibodywill realize the full potential of this extraordinary target and proveto be an invaluable therapeutic agent for the treatment of an extremelybroad spectrum of immune-related diseases wherein compounds thatantagonize CD40L/CD40 signaling may be used to intervene in the diseaseprocess.

That such antibodies would induce tolerance was not anticipated fromprior research. For example, previous studies in NHP using aglycosylatedαCD154 (αCD154^(agly)) antibodies that do not effectively bindcomplement or FcR have suggested that while the toxicities associatedwith αCD154 may have been eliminated, that such antibodies unfortunatelyeliminate the ability of the antibody to induce tolerance (Ferrant, J.L., Benjamin, C. D., Cutler, A. H., Kalled, S. L., Hsu, Y. M., Garber,E. A., Hess, D. M., Shapiro, R. I., Kenyon, N. S., Harlan, D. M., Kirk,A. D., Burkly, L. C. and Taylor, F. R., The contribution of Fc effectormechanisms in the efficacy of anti-CD154 immunotherapy depends on thenature of the immune challenge. Int Immunol 2004. 16: 1583-1594). Thisimpairment of functionality (tolerance induction) would suggest thatcomplement binding and activation is essential or at least important asto the ability of αCD154 to induce tolerance.

The prior studies which implicated complement binding to the efficacy ofan αCD154 antibody, and especially its ability to be therapeuticallyeffective such as inhibit the effects of CD154 in vivo, includingespecially the ability of the antibody to induce tolerance were effectedin complement deficient mice. While these studies and underlyingconclusions were deemed valid by other scientists, the subject inventorwanted to validate these studies in a different model because of thefact that complement deficient mice may not be the best model to studythe potential therapeutic efficacy of such antibodies. In particular, itwas theorized that this may not be the best model as these mice, becauseof their complement deficiency are defective in many aspects of immunityand also may express antibodies abnormally relative to wild-typeanimals, i.e., they may express grossly altered antibodies such asantibodies having aberrant or absent glycosylation (aglycosylated).Based on the potential effects of these abnormalities on the inductionof tolerance in vivo, the present inventor hypothesized that the priorstudies did not convincingly determine whether complement binding isindeed essential to the ability of an anti-CD40L antibody to inducetolerance, notwithstanding the conclusions of reputable scientistsinvolved in these studies.

More particularly, it was theorized that the reported results as to theinvolvement of complement in tolerance may be erroneous or overstated.Therefore, in order to critically assess the effects of complementbinding/activation and FcR binding on therapeutic efficacy and toxicitythe inventor elected to construct anti-CD154 antibodies containingmutations in their Fc region that preclude or prevent complement bindingand/or FcR binding. It was theorized that the toxicity of an anti-CD154containing a human constant region which results in thrombosis may beaddressed by introducing mutations which eliminate FcR binding, andpotentially that introducing such changes may not impact therapeuticefficacy such as the ability of the mutated antibody to inducetolerance, inhibit humoral and/or cellular immunity and to inhibit otheractivities elicited by CD154 and CD154/CD40 binding interactions.

In order to achieve these objectives the inventor desired to effectthese studies in an appropriate animal model. To meet these objectivesthe inventor, elected to conduct experiments in a rodent animal modelfor assaying thrombocytopenia and thrombosis which comprises andexpresses human FcRs. This animal model is disclosed in more detail inthe experimental examples infra.

As disclosed infra, the results of these experiments confirmed theinventor's hope, i.e., that anti-CD154 antibodies containing humanconstant regions such as IgG1 or IgG3 may be mutagenized at specificsites in their Fc regions in order to eliminate one or both of FcRbinding and/or complement binding to eliminate toxicity (thrombosis,clotting reactions), potentially the without loss of functionality.

Accordingly, based on the foregoing, mutated versions of antibodiescontaining mutated human constant regions, i.e., mutant IgG1 ((γ₁, γ₁^(−c), γ₁ ^(−FcR), γ₁ ^(−c/FcR)) which are specific to CD154 andcontaining mutations that disrupt complement binding and/or FcR bindingare disclosed herein.

In the working examples specific mutants were tested in order to assesswhether efficacy and toxicity of αCD154 are dependent on complementbinding (K322A, P331G, P331G/K322A) or FcR binding (N325L, K326V,E269R). Each of the complement binding engineered forms of αCD154 wastested for its ability to induce tolerance and the FcR binding mutantsfor their propensity to induce thromboembolic events in murine models.

While these specific mutations were exemplified, based on the resultsdisclosed infra it is anticipated that other recombinant anti-CD154antibodies may be developed comprising other Fc mutations reported todisrupt complement binding and/or FcR binding with similar properties,i.e./no toxicity and/or retained efficacy, including the ability toelicit tolerance in vivo. As discussed herein, other sites in the Fcregion of human IgG1 and other human constant regions are known to beinvolved in complement binding and/or activation as well as FcR binding.Accordingly, the described mutations are exemplary of appropriatemutations in the Fc region of IgG1 or IgG3 or other antibodies thatresult in loss of one or both of complement binding and FcR binding.

As further discussed in the examples, the tolerance inducing effects ofthe complement-binding mutant αCD154 variants were evaluated in awell-studied model of haplo-mismatched skin allograft survival, wherelong-term tolerance is induced by the administration of αCD154 andalloantigen. However, other tolerance models may alternatively be usedto assess the ability of a αCD154 complement deficient and/or FcRdeficient variant (containing a mutated Fc region) to induce tolerance.

The thromboembolic activities of the FcR mutated αCD154 is tested in amurine model expressing the human FcαRIIA receptor that reproduces theevents observed in NHP (Ferrant, J. L., Benjamin, C. D., Cutler, A. H.,Kalled, S. L., Hsu, Y. M., Garber, E. A., Hess, D. M., Shapiro, R. I.,Kenyon, N. S., Harlan, D. M., Kirk, A. D., Burkly, L. C. and Taylor, F.R., The contribution of Fc effector mechanisms in the efficacy ofanti-CD154 immunotherapy depends on the nature of the immune challenge.Int Immunol 2004. 16: 1583-1594). In such mice, treatment with αCD154(anti-mouse CD154) induces pulmonary thrombi; therefore we evaluatedtherein whether the loss of FcR binding in anti-mouse CD154 antibodiescontaining mutated human Fc regions (mutated IgG1 constant regions)eliminates or appreciably reduces the formation of thrombi. Thereby, theinventor can introduce specific Fc mutations, and based on the effectsof such antibodies containing these Fc mutations identify specificmutations that result in the eradication of the toxicity associated withαCD154 therapy. Thereupon, human, humanized or chimeric anti-human CD154antibodies, which elicit no toxicity but which retain efficacy, may beproduced containing such mutated human Fc regions.

As shown by the results in the experimental examples infra, the presentinventor has surprisingly shown, in contrast to the prior scientificconsensus that complement binding is not required for the ability of anαCD154 to induce T cell tolerance. Also, the results indicate that some,but not all mutations that reportedly impact FcR binding, eliminate thethromboembolic effects of αCD154. Based thereon, the invention providesa means for selecting antibodies containing appropriate Fc mutationsthat eliminate complement binding and/or FcR binding and which are wellsuited for use in anti-CD154 therapies such as described herein andwhich are not toxic.

In this regard, aside from the toxicity associated with FcR binding,because the subject antibodies may be mutated to eliminate complementbinding, the subject recombinant anti-CD154 antibodies are furtherbelieved to be less subject to toxicity concerns. This is advantageousbecause it has been reported that complement binding by some therapeuticantibodies may result in undesired toxicity in vivo. Accordingly, theinvention provides improved therapeutic anti-CD154 antibodies thatshould exhibit improved safety in human patients both from anelimination of thrombolytic adverse events attributable to FcR bindingand further by a reduction or elimination of complement associatedtoxicity. (See, e.g., “Complement Activation Plays a Key Role inAntibody-Induced Infusion Toxicity in Monkeys and Rats”, The Journal ofImmunology Feb. 15, 2008 vol. 180 no. 4 2294-2298).

The mutated CD154 specific antibodies of the present invention havingaltered complement and/or FcR binding may be used for the treatment andprevention of any condition wherein antagonizing the effects of CD154may be therapeutically effective, and may reduce the symptoms of thedisease. Examples thereof include the treatment of allergic, autoimmune,cancer, transplant, GVHD, inflammatory and other conditions, especiallyconditions wherein the induction of tolerance and/or the suppression ofhumoral immunity is therapeutically desirable. Specific examples arelisted in Table 1.

TABLE 1 Diseases and species that demonstrate efficacy of αCD154.Multiple sclerosis (EAE) Mouse, Human Rheumatoid arthritis MouseInflammatory bowel disease Mouse Thyroiditis Mouse Systemic LupusErythematosis Mouse, Human Autoimmune thrombocytopenia Human DiabetesMouse Graft vs. host disease Mouse Kidney transplantation Monkey Skintransplantation Mouse, Monkey BM transplantation Mouse AtherosclerosisMouse

The subject antibodies which target CD154, and which possess improvedsafety properties, are of great therapeutic potential as CD154 is anextremely attractive target for immune intervention in a wide spectrumof autoimmune, and graft-related diseases. Virtually all models ofautoimmune disease in mice (see Table 1) are therapeutically amelioratedby αCD154 treatment. Furthermore, the efficacy in mouse models hastranslated extremely well into man, as treatment of MS, Lupus and ITPall have documented efficacy of αhuman CD154 in clinical trials.

Beyond simply blocking CD154-CD40 interactions, αCD154 therapy leads tothe induction of immunologic tolerance (Prevention of transplantrejection by blocking CD40-CD154 interactions has been repeatedlydocumented for the induction of long-term tolerance to skin, Gordon, E.J., Markees, T. G., Phillips, N. E., Noelle, R. J., Shultz, L. D.,Mordes, J. P., Rossini, A. A. and Greiner, D. L., Prolonged survival ofrat islet and skin xenografts in mice treated with donor splenocytes andanti-CD154 monoclonal antibody. Diabetes 1998. 47: 1199-1206.; Markees,T. G., Phillips, N. E., Noelle, R. J., Shultz, L. D., Mordes, J. P.,Greiner, D. L. and Rossini, A. A., Prolonged survival of mouse skinallografts in recipients treated with donor splenocytes and antibody toCD40 ligand. Transplantation 1997. 64: 329-335; Jarvinen, L. Z., Blazar,B. R., Adeyi, O. A., Strom, T. B. and Noelle, R. J., CD154 on thesurface of CD4+CD25+ regulatory T cells contributes to skin transplanttolerance. Transplantation 2003. 76: 1375-1379; Quezada, S. A., Fuller,B., Jarvinen, L. Z., Gonzalez, M., Blazar, B. R., Rudensky, A. Y.,Strom, T. B. and Noelle, R. J., Mechanisms of donor-specific transfusiontolerance: preemptive induction of clonal T-cell exhaustion via indirectpresentation. Blood 2003. 102: 1920-1926; Frleta, D., Lin, J. T.,Quezada, S. A., Wade, T. K., Barth, R. J., Noelle, R. J. and Wade, W.F., Distinctive maturation of in vitro versus in vivo anti-CD40mAb-matured dendritic cells in mice. J Immunother 2003. 26: 72-84;Quezada, S., Eckert, M., Schned, A., Noelle, R. J. and Burns, C.,Distinct mechanisms of action of anti-CD154 in early versus latetreatment of murine lupus nephritis. Arth Rheum. 2003.; Elster, E. A.,Xu, H., Tadaki, D. K., Montgomery, S., Burkly, L. C., Berning, J. D.,Baumgartner, R. E., Cruzata, F., Marx, R., Harlan, D. M. and Kirk, A.D., Treatment with the humanized CD154-specific monoclonal antibody,hu5C8, prevents acute rejection of primary skin allografts in nonhumanprimates, Transplantation 2001. 72: 1473-1478., islets (Benda, B.,Ljunggren, H. G., Peach, R., Sandberg, J. O. and Korsgren, O.,Co-stimulatory molecules in islet xenotransplantation: CTLA4Ig treatmentin CD40 ligand-deficient mice. Cell transplantation 2002. 11: 715-720)bone marrow (Wekerle, T. and Sykes, M., Mixed chimerism andtransplantation tolerance. Annual review of medicine 2001. 52:353-370¹⁹, and a myriad of other transplanted organs (Camirand, G.,Caron, N. J., Turgeon, N. A., Rossini, A. A. and Tremblay, J. P.,Treatment with anti-CD154 antibody and donor-specific transfusionprevents acute rejection of myoblast transplantation. Transplantation2002. 73: 453-461; Tung, T. H., Mackinnon, S. E. and Mohanakumar, T.,Long-term limb allograft survival using anti-CD154 antibody in a murinemodel. Transplantation 2003. 75: 644-650). Furthermore, αhuman CD154 inNHP has been shown to induce long-term tolerance to allogeneic skintransplants.

Whereas anti-CD154 antibodies have been reported to have significanttherapeutic potential, the development of the inventive antibodiescontaining mutated Fc regions for use as therapeutics was not suggestedby the prior art as prior to the present invention it was thought thatC′ was involved (required) for mediating graft tolerance. Morespecifically it was thought that αCD154 must accomplish two things toinduce tolerance, prevent inflammation and activate C′. Surprisingly,this is not the case.

Quite surprisingly we teach herein that neither C′ activating activity,nor binding to FcR is necessary for an anti-CD154 antibody to betherapeutically effective (induce tolerance) and that antibodies whichcomprise specific mutations that eliminate or reduce FcR binding do notelicit thrombolytic or thrombocytopenia and therefore should beeffective and safe.

This was theorized in part because previous aglycosylated antibodiesthat have resulted in complete disabling of the Fc region of anti-CD154have eradicated toxicity, but substantially inhibited tolerance inducingefficacy. Only extremely high levels in mice (50 mg/kg×3) of Fc disabledanti-CD154 were shown to induce tolerance, but lower doses (20 mg/kg) inmonkeys could not induce tolerance.

By contrast, the inventor theorizes based on the results herein that C′activating activity is not required for the ability to induce toleranceand further theorize that binding to FcR may not be required for ananti-CD154 antibody to be therapeutically effective, and specifically toinduce tolerance or humoral suppression. Based on the foregoing, and theresults reported in the examples infra, the inventive anti-CD154antibodies which comprise specific mutations that eliminate or reduceFcR binding and which do not elicit thrombolytic or thrombocytopeniaand/or which further contain mutations in the Fc region that eliminateor reduce complement binding should be effective and moreover,substantially more safe than prior reported anti-CD154 antibodies,developed as potential human antibody therapeutics. In fact, to date, noanti-CD154 antibody has been approved for human therapy notwithstandingthe fact that the potential use of antibodies that target CD154 wasreported in the mid-90's and several such antibodies were in clinicaldevelopment.

Assessing αCD154 Toxicity in Mice

Earlier studies clearly documented the thrombogenic activities ofanti-CD154 mAbs in cynomolgus monkeys. In evaluating engineered forms ofanti-CD154 mAbs, studies in NHP is a costly and cumbersome approach.Therefore, less cumbersome and costly methods would be desirable such asassays using transgenic rodents.

It is believed that the binding of anti-C154-sCD154 (soluble (s) CD154is present in serum) immune complexes (IC) to platelets may be the basisfor the thrombogenic activity of anti-CD154 mAbs. Studies have shownthat anti-CD154 IC activate platelets in vitro via the IgG receptor(human FcγRIIA) (Langer, F., Ingersoll, S. B., Amirkhosravi, A., Meyer,T., Siddiqui, F. A., Ahmad, S., Walker, J. M., Amaya, M., Desai, H. andFrancis, J. L., The role of CD40 in CD40L- and antibody-mediatedplatelet activation. Thrombosis and homeostasis 2005. 93: 1137-1146) onplatelets and could cause thrombi formation. The prothrombotic effectsof anti-CD154 (using a human IgG1 variant of MR1) also have beenevaluated in vivo using hFcγRIIA transgenic mice (Robles-Carrillo, L.,Meyer, T., Hatfield, M., Desai, H., Davila, M., Langer, F., Amaya, M.,Garber, E., Francis, J. L., Hsu, Y. M. and Amirkhosravi, A., Anti-CD40Limmune complexes potently activate platelets in vitro and causethrombosis in FCGR2A transgenic mice. J Immunol 2010. 185: 1577-1583).These mice were produced because mice do not express FcγRIIA onplatelets. Upon injection of hIgG1/D154-sCD154 IC, mice developedpulmonary thrombi consisting of platelet aggregates and fibrin, similarto that observed in NHP treated with glycosylated anti-CD154 antibodies.By contrast, the administration of aglycosylated anti-CD154(hIgG1MR1agly) did not induce pulmonary thrombi. The inventoraccordingly elected to use this in vivo rodent assay to test theprothrombotic activity of different engineered human IgG₁ αCD154.

Therapeutic Applications of CD154 Antibodies of the Invention

As a category, there are nearly 50 million people in the US sufferingfrom the 100+ known autoimmune diseases. Treatment costs are estimatedto be over $100B/year and that figure is likely an underestimate. Costsfor the 7 major autoimmune diseases (IBD, Lupus, MS, RA, psoriasis andscleroderma) alone are estimated to range between $51-70.6 B/yr. In2008, there were 23, 288 transplants performed in the US. With anaverage cost of $22, 350/yr, over $500M/yr is spent on immunosuppressionpost-transplant.

αCD154 is potentially one of the most therapeutically valuable drugs forthe treatment of autoimmunity and graft rejection. In addition to thedemonstrated clinical efficacy seen in Lupus and ITP, the inventor wasinvolved in a completed a Phase I Clinical Trial in remitting/relapsing(RR) MS. While only a small cohort of patients was treated (12), theresults of the trial were striking. The conclusions of the trial werethat 4 weekly treatments with IDEC-131 resulted in: 1) No significantchanges in EDSS from baseline to 5 years for all doses; 2) Improved EDSScorrelated with increased dose and 3) Long-term follow up demonstrated aprofound reduction in clinical relapse rate that compares favorably tocurrent IMD. As a result of this trial, we were awarded an NIH grant toexecute a Phase II Clinical Trial in R/R MS but due to toxicityassociated with αCD154 seen in other trials, the αCD154 becameunavailable.

We theorized that if this toxicity problem can be resolved, and efficacysustained, that αCD154 is a viable and attractive therapeutic thatshould be permitted to re-enter human Trials. The subject improvedanti-CD154 antibodies may be used in any indication where interventionor blocking of the CD154 binding interaction is therapeuticallydesirable. These conditions include inflammatory, autoimmune, allergic,cancer, transplant, infectious, GVHD and other indications. The presentinventor will initially focus on developing anti-CD154 Abs for treatingMS and for commercial development as this is a devastating conditionthat affects many individuals, including many women in their late 20'sand 30's.

There is a wealth of data indicating that complement activation iscritical for the induction of tolerance by αCD154. Studies in complementdeficient mice clearly show that αCD154 is completely ineffective atinducing tolerance. While this has been interpreted as resulting fromcomplement-mediated elimination of activated T cells, the resultscontained in this application show that this is incorrect. Indeed, thepresent inventor initially thought that C′ activation at the cellsurface by αCD154 facilitates the generation of adaptive T^(reg) andexplained why aglycosylated αCD154 mAbs in NHP are ineffective atinducing tolerance. However, this invention reveals that to be erroneousas cannot be true as we teach herein the production of αCD154 mabs thatdo not bind C1q and yet still effectively induce tolerance. It isanticipated that such antibodies will be safe and effective and thatthese αCD154 mAbs useful for immune intervention.

Engineering Safe, Tolerance-Inducing αCD154.

To demonstrate efficacy and toxicity, a model antibody, MR1, waschimerized and engineered to eliminate or reduce complement binding(K322A, P331G, P331G/K322A) or FcR binding (N325L, K326V, E269R).Studies have shown that the human IgG1 version of MR1 is thrombogenic(Robles-Carrillo, L., Meyer, T., Hatfield, M., Desai, H., Davila, M.,Langer, F., Amaya, M., Garber, E., Francis, J. L., Hsu, Y. M. andAmirkhosravi, A., Anti-CD154 immune complexes potently activateplatelets in vitro and cause thrombosis in FCGR2A transgenic mice. JImmunol 2010. 185: 1577-1583) and that it can induce tolerance (Daley,S. R., Cobbold, S. P. and Waldmann, H., Fc-disabled anti-mouse CD40Lantibodies retain efficacy in promoting transplantation tolerance. Am JTransplant 2008. 8: 2265-2271).

We teach herein the synthesis of a chimeric hIgG1 form of MR1, which isengineered to contain well known mutations in the hIgG1 Fc regionreported to disrupt C1q binding or FcR binding. If this is shown to besafe and effective, i.e., eliminate thrombotic properties whilemaintaining tolerogenic properties this demonstrates that otheranti-CD154 antibodies, particularly those that bind human CD154 may besynthesized by engineering similar mutations that eliminate FcR bindingand optionally complement binding, which eliminate or reduce thrombosisor thrombocytopenia, while maintaining the antibody's ability to inducetolerance.

As disclosed in the working examples, the first step in engineering thehamster amurine CD154 into a human IgG1 is to clone and sequence the κand γ heavy chains. This has been accomplished and is presented in FIG.2 .

The generation and characterization of a series of Fc and C′ variants ofthe hIgG1 form of MR1 is then performed. Mutagenesis of residue 322 fromK→A (K322A) of IgG1 has been shown to abrogate complement activation. Ithas been shown that this variant binds human complement C1q with greatlylowered affinity and to inefficiently activate human C′ (Hessell, A. J.,Hangartner, L., Hunter, M., Havenith, C. E., Beurskens, F. J., Bakker,J. M., Lanigan, C. M., Landucci, G., Forthal, D. N., Parren, P. W.,Marx, P. A. and Burton, D. R., Fc receptor but not complement binding isimportant in antibody protection against HIV. Nature 2007. 449:101-104).

In addition the antibody was engineered in an effort to eliminate orreduce FcR binding in a manner that eliminates thrombotic or clottingtoxic reactions in vivo while not impacting its desired effects onimmunity such as tolerance. In this regard, there are sites reported toeliminate or reduce FcR binding. However, their effects on functionality(tolerance) and toxicity (thrombosis) of anti-CD154 antibodies wereuncertain.

For example, Shields R L, Namenuk A K, Hong K, et al. (High resolutionmapping of the binding site on Human IgG1 for Fc for FcγRI, Fc forFcγRII, Fc for FcγRIII, and FcRn) report the design of IgG1 variantswith impaired binding to the Fc for FcγR. J Biol. Chem 2001; 276:6591-604) In addition, some patents (US20070237767 and US20100104564)describe Fc mutagenesis to eliminate FcR binding.

Exemplary mutations reported to significantly reduce FcR binding aresummarized in Table 2, 3 and 4 below. Table 4 also identifies mutationsthat affect complement binding. Reported activities are reported asrelative folds comparing to the wild type Fc.

TABLE 2 Fc mutation FcγRI FcγRIIa FcγRIIb FcγRIIIa FcRn E233P 0.12 0.080.12 0.04 0.54 D265A 0.16 0.07 0.13 0.09 1.23 D265N 0.02 0.03 0.02 D270N0.03 0.05 0.04 N297A 0.15 0.05 0.1 0.03 0.8 S298N 0.05 0.08 0.06 P329A0.48 0.08 0.12 0.21 0.8 D270A 0.76 0.06 0.1 0.14 1.05

TABLE 3 Fc FcγRIIa FcγRIIa FcγRIIIa FcγIIIa mutation FcγRI (H131) (R131)FcγRIIb (V158) (F158) K326V 0.52 0.01 0.01 0.02 0.87 2.34 V369R 0.790.01 0.02 0.03 0.93 1.64 F405K 1.52 0.02 0.02 0.02 1.08 2.55 L410P 1.270.01 0.01 0.01 0.99 1.75 V427R 1.69 0.03 0.05 0.03 1.27 0.59

TABLE 4 Variant Fc mutation FcγRI FcγRIIa FcγRIIb FcγRIIc FcγRIIIa C1qFcRn 113 L234N 0.1 0.19 2.05 0.49 1.18 1.06 744 G237M 0.07 0.14 0.570.66 0.1 1.8 1.74 88 S239F 0.28 0.02 0.33 0.1 0.95 0.85 826 V262E 1.030.16 0.92 36.47 2.85 9.27 76 V264F 0.43 0.05 0.22 0.06 1.87 1.07 143V266T 0.28 0.1 0.16 0.18 1.21 0.53 228 S267N 0.72 0.08 0.27 3.18 0.85148 E269R 0.07 0.07 0.13 0.06 0.05 1.15 0.72 779 N286E 0.07 0.38 0.370.01 0 2.12 858 N297R 0.01 0.01 0.01 0.06 0.01 0.45 80 T299A 0.01 0.10.56 72.84 0.06 2.31 0.82 870 R301D 0.87 0.11 0.06 0.04 0.03 1.58 0.5 84N325L 0.42 0.04 1.46 0.03 2.18 0.91 161 N325E 1.34 0.09 0.05 0.03 <0.020.86 0.55 473 L328R 0.07 0.1 0.88 0.37 0.11 1.21 1.82

General Description of Inventive Methods

Preparation of MR1 Variants.

DNA encoding V_(H) and V_(L) of hamster amurine CD154 were cloned andfused to the human γ 1 C_(H)1, C_(H)2, C_(H)3 region or to describedvariants. The nucleotide sequences was verified using Megabace™ sequenceanalyzer. A plasmid expression vector, pEE12 containing both heavy andlight chains of each of the MR1 variants will be transfected into NS0cells and products purified by Protein A chromatography.

Binding to CD154.

Comparison of the binding activity of CD154 antibody variants wasdetermined by their binding to CHO cells transfected with mouse CD154.CD154-expressing CHO cells will be incubated with biotin-labeled αCD154in the presence of unlabeled αCD154 heavy chain variants orisotype-matched antibodies for 1 hr at 4° C. Binding of biotinylated MR1will be detected using a streptavidin conjugated fluorochrome and flowcytometry will be performed. The percent of inhibition by variants willbe deduced by recording reductions in the mean fluorescence intensity ofMR1 stained cells.

Antibody Half-Life Using ELISA

An αhuman IgG1 ELISA will be used to determine the half-life of all theIgG1 variants. Serum concentrations of hIgG1 will be determined over 1month post-administration.

Binding of Variants to FcRs.

Binding of each of the variant MR1 IgG1 mAbs to FcRs is determined by asolid phase assay. Briefly, Maxisorb ELISA plates will be coated withmouse or human FcγRI, FcγRIIA, FcγRIIB, or FcγRIIIA (R & D Systems). Wewill prepare biotinylated versions of the MR1 variants γ1 (WT), γ1^(−c)(K322A, P331G, P331G/K322A) and (K322A), γ1^(−FcR) (N325L, K326V,E269R). Other variants will have mutations in both the complement andFcR binding sites. Binding is determined by colorimetric detection usingenzyme-coupled avidin. Reduction in binding is determined for each ofthe variants compared to the WT γ1 molecule.

Binding of αCD154 mAbs to Human C1q

Purified human C1q will be titrated into wells in which the IgG1variants of MR1 have been absorbed onto Maxisorb ELISA plates. Bound C1qwill be detected with HRP-chicken anti-C1q. All variants will becompared to the binding of C1q to the WT IgG1 MR1, as described(Ferrant, J. L., Benjamin, C. D., Cutler, A. H., Kalled, S. L., Hsu, Y.M., Garber, E. A., Hess, D. M., Shapiro, R. I., Kenyon, N. S., Harlan,D. M., Kirk, A. D., Burkly, L. C. and Taylor, F. R., The contribution ofFc effector mechanisms in the efficacy of anti-CD154 immunotherapydepends on the nature of the immune challenge. Int Immunol 2004. 16:1583-1594.; and Taylor, P. A., Lees, C. J., Wilson, J. M., Ehrhardt, M.J., Campbell, M. T., Noelle, R. J. and Blazar, B. R., Combined effectsof calcineurin inhibitors or sirolimus with anti-CD40L mAb onalloengraftment under non-myeloablative conditions. Blood 2002. 100:3400-3407.

Induction of tolerance with mutant αCD154 mAbs. The hamster anti-murineCD154 that was produced in our laboratory 40 MR1 routinely induceslong-lived graft tolerance, as we have shown (Quezada, S. A., Fuller,B., Jarvinen, L. Z., Gonzalez, M., Blazar, B. R., Rudensky, A. Y.,Strom, T. B. and Noelle, R. J., Mechanisms of donor-specific transfusiontolerance: preemptive induction of clonal T-cell exhaustion via indirectpresentation. Blood 2003. 102: 1920-1926; Quezada, S. A., Bennett, K.,Blazar, B. R., Rudensky, A. Y., Sakaguchi, S. and Noelle, R. J.,Analysis of the underlying cellular mechanisms of anti-CD154-inducedgraft tolerance: the interplay of clonal anergy and immune regulation. JImmunol 2005. 175: 771-779; Rossini, A. A., Parker, D. C., Phillips, N.E., Durie, F. H., Noelle, R. J., Mordes, J. P. and Greiner, D. L.,Induction of immunological tolerance to islet allografts. CellTransplant 1996. 5: 49-52). Tolerance is induced by theco-administration of alloantigen (in the form of donor spleen cells) andαCD154. It has been shown that a humanized IgG1 form of MR1 also inducesgraft tolerance 24, and therefore the WT al variant will serve as apositive control for tolerance induction. The mutant versions of MR1which have lost the ability to bind complement will be tested for theirability to induce graft tolerance.

Skin grafting is performed as a modification of a technique used byMarkees et al. (See reference citation 12). Briefly, age-matched maleCB6F1 mice will be used as donors for both spleen cells (DST) and skingrafts. Recipient C57BL/6 mice will injected with or without 5×107 DSTcells in 500 μL Hanks balanced salt solution by tail vein injection(intravenously) and 500 μg of WT or mutant αCD154 or controlimmunoglobulin, hamster or human, (HIgG1) in phosphate-buffered saline(PBS) intraperitoneally on days −3, −5 and −7. Mice will treated withthe appropriate antibody (250 ag/injection) 3 times per week, thereafterfor the duration of the experiment. On day 0, recipient mice will beanesthetized with 50 μg per gram body weight of each of ketamine andxylazine injected intraperitoneally (15 mg/mL in PBS), and CB6F1 skingrafts will be prepared using established methods. Rejection will bedefined as the day on which less than 20% of the skin graft remains.Animals will be evaluated for skin graft rejection for 100 days. Inaddition, for each of the tolerant groups, skin grafts will be taken atday 100 and evaluated by histochemistry for leukocyte infiltrates andscored based on the number of cells/area measured. Finally, third partytransplants (H-2Kskin) will be transplanted on tolerized mice (inselected groups) to assure that the tolerance induced is antigenspecific, as has been published previously in this system (Markees, T.G., Phillips, N. E., Noelle, R. J., Shultz, L. D., Mordes, J. P.,Greiner, D. L. and Rossini, A. A., Prolonged survival of mouse skinallografts in recipients treated with donor splenocytes and antibody toCD40 ligand. Transplantation 1997. 64: 329-335, Markees, T., Phillips,N., Gordon, E., Noelle, R. J., Mordes, J. P., Greiner, D. L. andRossini, A. A., Improved skin allograft tolerance induced by treatmentwith donor splenocytes and an extended course of anti-CD154 monoclonalantibody. Transplant Proc 1998. 30: 2444-2446; Markees, T. G., Appel, M.C., Noelle, R. J., Mordes, J. P., Greiner, D. L. and Rossini, A. A.,Tolerance to islet xenografts induced by dual manipulation of antigenpresentation and co-stimulation. Transplantation Proceedings 1996. 28:814-815) of humoral immunity with mutant αCD154 mAbs.

In addition to measuring the impact of the complement binding mutants(K322A, P331G, P331G/K322A) on tolerance, we also will measure theimpact of antibody treatment on the development of primary and secondaryhumoral immune responses, as previously described. Briefly, mice(4/group) will be immunized with chicken ovalbumin in CFA (200 ag/mouse)and treated with the MR1 variants (200 ag/mouse×3 times/week). On days7, 14 and 21, IgM and IgG anti-OVA will be measured by a standardizedanti-OVA ELISA and serum concentrations of anti-OVA will be quantified.It is anticipated that such variants will be effective at inhibitinghumoral immunity.

Toxicity Studies with Mutant αCD154 mAbs.

The thrombogenic activity of αCD154 has been demonstrated in a murinemodel using mice that express human FcγRIIA. This model parallelstoxicity findings in NHP using both intact and aglycosylated forms ofanti-human CD154. Briefly, mice will be injected with preformed immunecomplexes (IC) of sCD154 (R & D Systems) and each variant of αCD154 (138μg mAb and 50 μg Ag, approximating 500 nM IC at a 1:3 (mAb/Ag)stoichiometric ratio). Following injection, if the mixture isthrombolytic, mice will exhibit prolonged disorientation, shallowbreathing, and impaired mobility. Those exhibiting this activity areexpected to have marked reductions in platelet counts. After 60 minutes,lungs will be harvested, fixed in formalin, sectioned and H&E-stained.Mouse lung sections will be evaluated for evidence of thrombosis (asmeasured by intravascular thrombi) and the number of thrombi/sectionwill be counted. For each mouse, 10 sections will be counted and thetotal number of thrombi compared across all groups treated with thevarious variants of IgG1 MR1. In addition, total platelet counts(harvested by cardiac puncture at the time of euthanasia), will beevaluated by flow cytometry, and are expected to drop by 80% using thoseantibodies that are thrombogenic. These findings will determine which ofthe MR1 variants (FcR binding (N325L, K326V, E269R) are thrombogenic andif alteration of the FcR binding alters this activity.

Blocking the Development of a T Cell Mediated Autoimmune Disease,Experimental Autoimmune Encephalomyelitis (EAE).

It has not been reported that C′ activation is critical for anti-CD154induced protection in EAE. Our data show that short-term interventionleads to long term remission, which suggests that it induces tolerance.It has been reported that MR1^(aglys) inhibits EAE49, however, thisspecific mAb only had a 50% reduction in C1q. As we have extensiveexperience in anti-CD154 in treatment of EAE (we will evaluate each ofthe variants in this disease model) to address the potential of each inblocking cell-mediated immunity.

Female C57BL/6 mice 5-8 weeks old will be immunized subcutaneously with200 μg of MOG35-55 peptide emulsified in CFA supplemented with 5 mg/mlof Mycobacterium tuberculosis. The mice will receive intraperitonealinjections with 250 ng pertussis toxin at the time of immunization and48 hours later. After 7 days, the mice will receive an identical boosterimmunization with MOG/CFA without pertussis toxin. Clinical diseaseusually commences between day 16 and day 20 after immunization. Micewill be administered each of the MR1 variants, human IgG (as control forthe variants), hamster Ig (as control for MR1) or hamster MR1 (200μg/mouse 3×/week) for the duration of the experiment (50 days).

Clinical evaluation. Mice will be scored four times per week as follows:0, no detectable signs of EAE; 0.5, limp distal tail; 1, complete limptail; 1.5, limp tail and hind limb weakness; 2, unilateral partial hindlimb paralysis; 2.5, bilateral partial hind limb paralysis; 3, completebilateral hind limb paralysis; 3.5, complete hind limb paralysis andunilateral forelimb paralysis; 4, total paralysis of both forelimbs andhind limbs; 5, death. Mice scoring greater than 4 but less than 5 willbe euthanized.

Determination of Toxicity

A desired antibody according to the invention will have greatly reducedor no toxicity in the disclosed thrombotic animal model.

Determination of Efficacy

Efficacy (induction of tolerance) will be assessed in the disclosed skingraft model of tolerance.

The following examples illustrate the operability of the invention indeveloping safe and improved, functionally active anti-CD154 antibodiesfor use in immune therapies.

Example 1: Design of Anti-CD154 Antibodies with Impaired Fc R BindingActivities and Functional Properties

Assessment of the Capacity of MR1 and MR1-Derived Monoclonal Anti-MouseCD154 Antibodies to Activate Platelets in Mice Transgenic for HumanFcγRIIA

As discussed herein, in early clinical trials, it was reported that mAbstargeting CD154, which is important in autoimmune and other diseases,displayed an unexpected association with thrombosis (induced blood clotswhich may cause death or stroke). The mechanisms by which such mAbs areapparently associated with thrombosis were unknown, in part because thedisease conditions in which they were used are independently associatedwith thrombosis. Additionally, there is no known molecular mechanism bywhich antibodies directly activate coagulation (i.e., the blood clottingsystem that drives thrombosis); hence, one or more componentsintermediary between therapeutic mAbs and coagulation per se must beinvolved. In the case of heparin-induced thrombocytopenia (HIT), asingle intermediary component has been identified: the platelet IgGreceptor, FcγRIIa.

HIT is a drug-induced thrombotic autoimmune syndrome in which IgGantibodies can induce a thrombotic state in patients—not by directlyactivating coagulation, but rather by forming immune complexes (ICs)with a platelet antigen target, PF4 (bound to the drug, heparin), andsubsequently activating platelet FcγRIIa, which leads to multipleplatelet-dependent prothrombotic processes, including coagulationactivation and thrombosis. Attempts to replicate HIT's thromboticprocesses in a mouse model were hindered by the fact that mice lack theequivalent of the human FcγRIIA gene. McKenzie and colleagues thus mademice transgenic for human FcγRIIa (FCGR2A mice) and went on todemonstrate that the HIT thrombotic phenotype could be fully replicatedin FCGR2A mice, but not in mice lacking this IgG receptor McKenzie S E,Taylor S M, Malladi P, Yuhan H, Cassel D L, Chien P, Schwartz E,Schreiber A D, Surrey S, Reilly M P. The role of the human Fc receptorFc gamma RIIA in the immune clearance of platelets: a transgenic mousemodel. J Immunol. 1999; 162:4311-8).

It was later shown that anti-CD154 mAbs, when combined with CD154 (humanor mouse), rapidly induced thrombocytopenia and thrombosis in FCGR2A,but not wild type (WT) mice. (Robles-Carrillo L, Meyer T, Hatfield M,Desai H, Dávila M, Langer F, Amaya M, Garber E, Francis J L, Hsu Y M,Amirkhosravi A. Anti-CD154 immune complexes potently activate plateletsin vitro and cause thrombosis in FCGR2A transgenic mice. J Immunol.2010; 185:1577-83). These studies suggested that any therapeutic mAbassociated with thrombosis may depend, at least in part, on theactivation of the platelet IgG receptor. It will thus be informative toevaluate the platelet-activating capacity of anti-CD154 mAbs beingdeveloped for therapeutic uses by treating FCGR2A mice with such mAbs,and subsequently identifying how this affects, if at all, the onset ofthrombocytopenia or thrombosis. Such testing will be particularly usefulfor anti-CD154 mAbs that have been engineered to have reduced capacityfor triggering FcγRIIa-dependent platelet activation.

It has been reported that a humanized form of MR1, when combined withits antigen target, mouse CD154, rapidly induced severe thrombocytopenia(loss of circulating platelets) and pulmonary thrombosis in FCGR2A mice.(Robles-Carrillo L, Meyer T, Hatfield M, Desai H, Dávila M, Langer F,Amaya M, Garber E, Francis J L, Hsu Y M, Amirkhosravi A. Anti-CD154immune complexes potently activate platelets in vitro and causethrombosis in FCGR2A transgenic mice. J Immunol. 2010; 185:1577-83) Inthis same study, an aglycosylated humanized anti-mouse CD154 mAb, MR1,which is presumed to have greatly reduced capacity to activate FcγRIIa,did not induce thrombocytopenia or thrombosis.

In the experiments herein, we tested variants of monoclonal anti-mouseCD154, MR1, and derivatives thereof, in the above-described FCGR2A mousemodel of thrombosis. The specific aim of this study was to inject FCGR2Amice with preformed ICs consisting of mouse CD154 plus MR1 or variousMR1 derivatives and to identify: (1) any possible evidence ofthrombocytopenia, (2) any possible evidence of pulmonary thrombosis, and(3) any possible behavioral signs of thrombotic stress subsequent toIC-induced platelet activation.

Materials and Methods

Materials:

Four anti-mouse CD154 antibodies were tested in FCGR2A mice:

PBS (baseline controls used for comparison with test mAbs, below)

MR1-WT (a humanized MR1 anti-mouse CD154 mAb)

N325L (a variant of MR1-WT)

K326V (a variant of MR1-WT)

E269R (a variant of MR1-WT)

Murine soluble CD154 (or “sCD154”) was purchased from Peprotech, Inc.(Rocky Hill, N.J.).

Methods:

Preparation and delivery of immune complexes (IC): Mouse sCD154 (60 μg)was combined with anti-CD154 mAb (175 μg) in PBS to prepare 250 μlvolume of mCD154+anti-CD154 IC solution, 200 μL of which was injectedintravenously into each FCGR2A mouse within 5 minutes of IC preparation.

Experimental Animals:

Twenty four FCGR2A mice (8-12 week old, male or female) mice weredivided into five groups (one per test mAb, and one PBS negativecontrol) of six animals per group. The genotype of all FCGR2A animalsused in the study were verified by PCR as per Jax Labs protocol.

Intravenous Injection of IC:

Unanesthetized mice were restrained in a standard mouse restrainer. Thelateral tail vein was dilated by warming with a heat lamp. IC solutionswere then injected slowly (˜10 seconds), and mice were transferredimmediately to an empty cage for observation.

Observation of Symptoms:

Following IC injection, each mouse was continuously monitored inisolation for ten minutes. During this period, observers assessed andrecorded the mice's locomotion, gait, breathing, and monitored the micefor signs of thrombotic stress (such as disorientation and partial ortemporary paralysis). Four categories were used to summarize the complexof symptoms observed in test animals: (1) None—no abnormalities inlocomotion, gait, breathing, and no sign of disorientation or paralysis;(2) Mild—no sign of disorientation or paralysis, normal locomotion, butsigns of lethargy and rapid breathing; (3) Moderate—lethargy, rapidbreathing, disruption of locomotion except following contact byobserver; (4) Severe—disorientation, signs of paralysis or completeimmobility.

Blood Collection and Platelet Counting:

Ten minutes after IC injection, mice were anesthetized by isoflurane andapproximately 500 μl of blood was collected into citrate anticoagulantby cardiac puncture using a 25 gauge needle. Platelet counts weredetermined electronically using an Coulter Act diff Counter within 2minutes of blood collection. Platelet counts were adjusted for thevolume of citrate in the collection tube and recorded for each animal.

Assessment of Thrombosis in the Pulmonary Vasculature:

Immediately after blood draw, entire lungs were dissected, rinsed in PBSbuffer, and placed in buffered formalin. Twenty four hours later,paraffin blocks were prepared and 3 m slide sections were cut andstained with hematoxylin and eosin (H&E) for histological evaluation forthe presence of thrombi. Five slides were prepared from the mid-organregion of each lung with spacing between cut section of approximately50-100 μm. Each slide was assessed by two independent observers blindedto the identity of the animal groups from which the slides wereprepared. Five randomly chosen fields were assessed per slide. In caseswhere greater than 9 thrombi were observed per field, no attempt wasmade to determine the precise number of thrombi, and the value of 10(ten) was entered as the nominal observation.

Statistical Analysis:

Data were analyzed by SigmaPlot . . . . Platelet counts and number ofclots/field between groups were analyzed using the Kruskal-Wallis OneWay Analysis of Variance on Ranks

Results

The first group of animals were injected with PBS (200 μL delivered) inorder to obtain baseline platelet counts and normal lungs forhistological analysis. These values are compared below to test animalgroups. Following PBS injection, all animals exhibited normallocomotion, gait, breathing, and showed no signs of thrombotic stress(such as disorientation and partial or temporary paralysis). Animalsinjected with MR1-WT mAb showed signs of moderate to severe signs ofthrombotic stress (FIG. 6 ), which correlated with loss of circulatingplatelets (FIG. 7 ), and histologic observation of the prevalence ofpulmonary thrombi (FIG. 8 ). The injection of animals with N325L andK326V mAbs gave similar results (did not prevent thrombosis). In manycases, histologic evidence of thrombosis greatly exceeded 10 clots perfield. All animals injected with E269R mAb exhibited normal locomotion,gait, breathing, and showed no signs of thrombotic stress. The lungvasculature of all E269R-injected mice were free of thrombi. (Seehistologic data also in FIGS. 9-13 )

It should be noted that two of six mice injected with N325L did notexperience thrombotic thrombocytopenia. The causes of these anomaliesare unknown; however, in our experience, such occasional outliers canoccur in experiments of this type. On the other hand, because theplatelet counts correlated with the relative absence of pulmonarythrombi from these two mice, the data were included in the statisticalanalysis comparing the experimental groups.

Conclusions

In this mouse model of antibody-induced thrombocytopenia and thrombosis,MR1-WT, N325L, and K326V demonstrated potent activity, whereas E269Rlacked activity and was comparable by all measures with the PBS negativecontrol group.

Example 2: Design of Anti-CD154 Antibodies with Impaired ComplementDependent Cytotoxicity (CDC) Activities and Functional Properties

Cloning and Synthesis of Chimeric Anti-CD154 Antibody (MR1) with HumanIgG1 Constant Regions

It was initially theorized by the present inventor, in part based onprior literature, that anti-CD154 antibodies lose their ability toinduce tolerance when the C1q binding site is mutated Based thereon, weassumed that a model anti-CD154 antibody, i.e., the murine anti-CD154(MR1) having the variable heavy and light sequences in FIG. 2 would loseits ability to induce tolerance when the C1q binding site is mutated.

To this end, MR1 was converted into a human IgG1. It has previouslyreported that a human IgG1 version of MR1 can induce tolerance (Daley,S. R., Cobbold, S. P. & Waldmann, H. Fc-disabled anti-mouse CD154antibodies retain efficacy in promoting transplantation tolerance. Am JTransplant 8, 2265-2271 (2008)). As described below a chimeric hIgG1form of MR1 was produced and then engineered to introduce mutations inthe hIgG1 Fc region that disrupt C1q binding.

The first step in engineering the hamster anti-murine CD154 into a humanIgG1 (MR1 hIgG1) is to clone and sequence the light and heavy chains ofMR1. DNA encoding VH and VL of hamster anti-CD154 MR1 have been clonedand fused to the human γ 1 CH1, CH2, CH3 region or to variants describedbelow. The nucleotide sequences have been verified using Megabace™sequence analyzer and are shown in FIG. 2 . A plasmid expression vector,pEE12 containing both light and heavy chains of each of the MR1 variantswas transfected into NS0 cells and products purified by Protein Achromatography.

The generation and characterization of a series of C′ variants of thehIgG1 form of MR1 was then effected.

Designing Fc Variants with Impaired CDC.

No single or combinations of Fc mutations have been reported to ablatethe CDC activity while maintaining near wild type ADCC activity.However, CDC assay conditions may effect this analysis. For example, CDCactivities can differ significantly depending on target cells, dilutionfactors of the complement, and species sources of the complement whichcould be from human, guinea pig, or rabbit as well as other factors.Given our analysis we believe that the best single and double mutationcandidates for impaired CDC activity without significant effects on ADCCare: K322A, P331G, and P331/K322A.

Mutagenesis of K322A and P331G of IgG1 have been shown to abrogatecomplement activation. It has been shown that this variant binds humancomplement C1q with greatly lowered affinity and inefficiently activateshuman C′. (Hessell, A. J., et al. Fc receptor but not complement bindingis important in antibody protection against HIV. Nature 449, 101-104(2007)).

Measurement of Loss of C1q Binding by MR1 hIgG1 Mutants.

The binding of C1q to each of the MR1 hIgG1 mutants was evaluated. Formeasuring C1q binding to MR1, purified MR1 variant antibody (AragenBioscience, Morgan Hill, Calif.), was diluted to 100, 10, 1 and 0.1μg/ml in phosphate-buffered saline (PBS) to coat a 96-well enzyme-linkedimmunosorbent assay (ELISA)-grade plate (ThermoScientific, Florence,Ky.) overnight at 4° C. The plate was then washed three times withPBS-0.05% Tween 20 (Tw20) and blocked for 1 h with 1% bovine serumalbumin (BSA)-Tw20-PBS at room temperature. Complement component C1qfrom human serum (Sigma, St. Louis, Mo.), was diluted to 1 μg/ml in 1%BSA-Tw20-PBS then plated and allowed to incubate for 1 h at roomtemperature. The plate was washed three times with PBS-Tw20, andhorseradish peroxidase-labeled sheep anti-human C1q (GenWay Biotech, SanDiego, Calif.) was added. After a 1 h, room temperature incubation, theplate was washed three times with PBS-Tw20, then TMB(3,3′,5,5′-tetramethylbenzidine) (ThermoScientific) provided acolorimetric change which was then quantitated at 450 nm by an ELISAreader (BioTek, Winooski, Vt.).

The results of these experiments are in FIG. 4 . As shown therein, allof the mutants had reduced C1q binding.

Functional Studies with Mutant Anti-CD154 Mabs.

The hamster anti-murine CD154 that was produced in our laboratory 3MR1routinely induces long-lived graft tolerance, as we have shown (Noelle,R. J., et al. A novel ligand on activated T helper cells binds CD40 andtransduces the signal for the cognate activation of B cells. Proc. Natl.Acad. Sci. USA89, 6550-6554 (1992)).

However, previous reports have suggested that in complement deficientanimals that anti-CD154 antibodies do not elicit tolerance. (Quezada, S.A., et al. Analysis of the underlying cellular mechanisms ofanti-CD154-induced graft tolerance: the interplay of clonal anergy andimmune regulation. J Immunol 175, 771-779 (2005); Quezada, S. A., et al.Mechanisms of donor-specific transfusion tolerance: preemptive inductionof clonal T-cell exhaustion via indirect presentation. Blood 102,1920-1926 (2003); Quezada, S. A., Jarvinen, L. Z., Lind, E. F. & Noelle,R. J. CD40/CD154 Interactions at the Interface of Tolerance andImmunity. Annu Rev Immunol 22, 307-328 (2004); Rossini, A. A., et al.Induction of immunological tolerance to islet allografts. CellTransplant 5, 49-52 (1996).) Therefore, we assessed whether our mutantswere able to elicit tolerance. In these experiments, the four hIgG1versions of MR1 (MR1 WT, K322A, P331G, and P331/K322A) were tested fortheir ability to induce graft tolerance and the results of theseexperiments are in FIG. 5 .

Skin grafting was effected using a modification of the techniquedeveloped by Markees et al. (Markees, T. G., et al. Prolonged survivalof mouse skin allografts in recipients treated with donor splenocytesand antibody to CD40 ligand. Transplantation 64, 329-335 (1997)).Briefly, age-matched male CB6F₁ mice were used as donors of both spleencells (DST) and skin grafts. More specifically, Tail skin (μl cm2) fromCB6F1 (F1) female donors was transplanted onto the dorsal area ofage-matched C57BL/6 females. To induce T cell tolerance, recipientsreceived T-depleted spleen cells (DST) by IV tail injection from F1donors on day −7 before skin graft (day 0) and 200 μg of MR-1 variantsIP on days −7, −5, and −3. Grafts were observed 3 times per weekstarting on day 8. Grafts were considered rejected when 80% of theoriginal graft disappeared or became necrotic.

Recipient mice were injected with or without 5×10⁷ DST cells in 500 μLHanks balanced salt solution by tail vein injection (intravenously) and500 μg of anti-CD154 or control hamster immunoglobulin (H-Ig) inphosphate-buffered saline (PBS) intraperitoneally. Mice were injectedwith the MR1 variants or H-Ig 3 times per week for the duration of theexperiment. On day 0, recipient mice were anesthetized with 50 μg pergram body weight of each of ketamine and xylazine injectedintraperitoneally (15 mg/mL in PBS), and CB6F₁ or C57BL/6 skin graftswere prepared using established methods. Rejection was defined as theday on which less than 20% of the skin graft remained.

Afterward the results were analyzed. Treatment with control human IgG1and DST did not prolong rejection, as was anticipated. However, as canbe seen in FIG. 6 , like WT H IgG1 MR1, all of the mutant MR1 antibodiesinduced long lived graft acceptance. Hence, C1q binding and complementactivation by anti-CD154 antibodies IS NOT essential to induce grafttolerance. This is in contrast to what was observed in the complementdeficient mice, and suggests that the complement deficient mice likelyhave some other anomalies that influence the induction of grafttolerance. Thus, therapeutically effective αCD154 antibodies can beproduced that do not bind complement.

Example 3: Expression and Binding Properties of Anti-Human CD154Antibodies with Impaired Complement and FcR Binding Properties

Two anti-human CD40L IgG1 antibodies each comprising the variable heavyand light sequences in FIG. 20 and modifications in the human IgG1constant region were expressed in a mammalian cell line (HEK-293Freestyle cell line). The first antibody, Antibody A, contains V215A andE269R mutations. Antibody B contains 3 mutations in the constant region,i.e., V215A, E269R and K322A mutations.

Both antibodies were purified by conventional methods and the bindingactivity of both recombinantly expressed antibodies to hCD40L wasassayed. To measure anti-hCD40L variants binding to hCD40L, purifiedrecombinant human CD40L was bound to a 96-well ELISA-grade plate. Afterblocking, anti-hCD40L variants were added followed by mouse anti-humanIgG then horseradish peroxidase-labeled goat anti-mouse IgG. TMB colordevelopment was then quantitated at 450 nm by an ELISA reader.

As evidenced by the ELISA binding data contained in FIG. 19 , bothAntibody A and B specifically bind to hCD40L. Based on the informationin this application these antibodies should, be safe and effective,i.e., not elicit an adverse thrombogenic reaction, when used in humantherapies wherein antagonizing CD40L and/or the CD40L/CD40 interactionis therapeutically or prophylactically beneficial.

REFERENCES CITED IN APPLICATION

The following references are cited. The contents of all are incorporatedby reference herein.

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The invention claimed is:
 1. A nucleic acid encoding a humanizedanti-CD154 antibody of the human IgG1 isotype, comprising an IgG1 Fcregion which comprises an E269R mutation and a K322A mutation and whichfurther comprises one additional Fc modification which impairs FcRbinding and/or impairs complement binding, wherein said anti-CD154antibody comprises the V_(L) complementarity determining regions (CDRs)of SEQ ID NO: 17, 18, and 19 and the V_(H) CDRs of SEQ ID NO: 24, 25 and26 wherein the one additional Fc modification which impairs FcR bindingis selected from the following mutations: V215A, E233P, D265A, D265N,D270N, N297A, S298N, P329A, D270A, K326V, V369R, F405K, L410P, V427R,L234N, G237M, S239F, V262E, V264F, V266T, S267N, N268E, N297R, T299A,R301D, N325L, N325E and L328R, and the additional Fc modification whichimpairs complement binding comprises a P331G or P331G/K322A mutation. 2.An expression vector comprising the nucleic acid of claim 1 operablylinked to a promoter.
 3. A recombinant cell comprising the nucleic acidof claim 1 operably linked to a promoter.
 4. A method of producing ahumanized anti-CD154 antibody comprising culturing a cell comprising thenucleic acid of claim 1 operably linked to a promoter under conditionsthat permit expression of the humanized anti-CD154 antibody encoded bysaid nucleic acid.
 5. The method of 4, which comprises isolating theexpressed humanized anti-CD154 antibody from the cell or culture mediumcomprising the cell.
 6. A nucleic acid encoding a humanized anti-CD154antibody of the human IgG1 isotype, comprising an IgG1 Fc region whichcomprises an E269R mutation and a K322A mutation.
 7. The nucleic acid ofclaim 6, wherein the IgG1 Fc region further comprises a V215A mutation.8. An expression vector comprising the nucleic acid of claim 6 operablylinked to a promoter.
 9. A recombinant cell comprising the nucleic acidof claim 6 operably linked to a promoter.
 10. A method of producing ahumanized anti-CD154 antibody comprising culturing a cell comprising thenucleic acid of claim 6 operably linked to a promoter under conditionsthat permit expression of the humanized anti-CD154 antibody encoded bysaid nucleic acid.
 11. The method of 10, which comprises isolating theexpressed humanized anti-CD154 antibody from the cell or culture mediumcomprising the cell.